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| The EMBO Journal
(2001) 20, 3928–3937, doi:10.1093/emboj/20.15.3928 |
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| Mutations lowering the phosphatase activity of HPr kinase/phosphatase switch off carbon metabolism |
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| Vicente Monedero1, Sandrine Poncet1, Ivan Mijakovic1, Sonia Fieulaine2, Valérie Dossonnet1, Isabelle Martin-Verstraete3, Sylvie Nessler2 and Josef Deutscher1 |
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1 Laboratoire de Génétique des Microorganismes, INRA and CNRS URA1925, Thiverval-Grignon, France 2 Laboratoire d'Enzymologie et Biochimie Structurales, CNRS UPR9063, Gif sur Yvette, France 3 Institut Pasteur, Unité de Régulation de l'Expression Génétique, CNRS URA2171, France To whom correspondence should be addressed Josef Deutscher, jdeu@grignon.inra.fr Received 14 November 2000; Revised 1 March 2001; Accepted 12 June 2001. |
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| Abstract |
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| The oligomeric bifunctional HPr kinase/P-Ser-HPr phosphatase (HprK/P) regulates many metabolic functions in Gram-positive bacteria by phosphorylating the phosphocarrier protein HPr at Ser46. We isolated Lactobacillus casei hprK alleles encoding mutant HprK/Ps exhibiting strongly reduced phosphatase, but almost normal kinase activity. Two mutations affected the Walker motif A of HprK/P and four a conserved C-terminal region in contact with the ATP-binding site of an adjacent subunit in the hexamer. Kinase and phosphatase activity appeared to be closely associated and linked to the Walker motif A, but dephosphorylation of seryl-phosphorylated HPr (P-Ser-HPr) is not simply a reversal of the kinase reaction. When the hprKV267F allele was expressed in Bacillus subtilis, the strongly reduced phosphatase activity of the mutant enzyme led to increased amounts of P-Ser-HPr. The hprK V267F mutant was unable to grow on carbohydrates transported by the phosphoenolpyruvate:glycose phosphotransferase system (PTS) and on most non-PTS carbohydrates. Disrupting ccpA relieved the growth defect only on non-PTS sugars, whereas replacing Ser46 in HPr with alanine also restored growth on PTS substrates. |
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| Keywords: bifunctional enzymes, carbohydrate metabolism, HPr, HPr kinase:P-Ser-HPr phosphatase, PEP:glycose phosphotransferase system |
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